Nuclear pore complexes (NPCs) are molecular sorting machines that ensure proper compartmentalization of nuclear and cytoplasmic contents in all eukaryotes. Ions, metabolites and small macromolecules pass freely across the NPC, whereas the translocation of larger (>40kD) macromolecules is impeded. Simultaneously with this barrier function, the NPC exhibits remarkable selectivity towards a group of highly mobile nuclear transport receptors (NTRs); NTRs bind signal-bearing cargo molecules and facilitate their import/export through the NPC. These selective transport events are extremely rapid and an individual NPC can transport 1000 molecules/second. How the NPC establishes this selective and efficient transport system is not completely elucidated and represents a fundamental challenge to our understanding of cellular compartmentalization. Moreover, understanding the function of the NPC will be critical for designing strategies to ameliorate a growing number of human pathologies including cancers, heart abnormalities, neurodegenerative diseases, viral infection, in addition to developmental defects that result when NPC function is perturbed. Lastly, at its core, the NPC is an efficient molecular sorting machine - defining the mechanism of transport will lead to the development of synthetic materials that mimic its properties for protein purification, biotechnology and pharmaceutical applications, including bioreactors. The molecular basis for NPC selectivity and rapid transport are interactions between NTRs and a subset of NPC proteins (nups) that are rich in phenylalanine-glycine (FG) amino acid residues. However, measured affinities between NTRs and FG-nups are too strong to support observed in vivo transport rates leading to a paradox; we (and others) suggest that this paradox reflects the limitations of examining individual FG-nups outside of their native environment, where the presence of other FG-nups with different properties, their stoichiometry, and their confinement within a cylindrical channel cumulatively contribute to a cooperative behavior that is difficult to recapitulate in vitro. By leveraging our expertise in DNA-origami we have the ability to fabricate structures termed NuPODs (Nuclear Pore Complex Organized by DNA) that mimic the dimensions of the NPC and contain defined compositions of FG-nups that are precisely spatially arranged. In Aim 1, we will use these NuPODs as binding supports to directly test how spatial-positioning and FG-density impact the cooperative behavior of FG-nups and their binding kinetics to specific NTRs. In Aim 2, we will immobilize our NuPODs on nanopores and examine how unique combinations of FG-nups establish a permeability barrier to inert macromolecules of varying sizes. These studies will open the door for the fabrication of NPC-mimics that fully recapitulate the transport properties of the NPC. Further, our understanding of the NPCs underlying design will allow us to generate NuPODs with prescribed selectivity/permeability characteristics.